Heparin is a highly sulfated glycosaminoglycan (GAG) consisting of repeating disaccharide units with an average molecular weight of 12-15 kilodaltons (kD). Most heparin prepared commercially for infusion into human patients is isolated from the gut mucosa of pigs in the form termed “unfractionated heparin” (UFH). Fractionation of UFH by various methods results in low molecular weight heparin (LMWH) with 60% of the polysaccharide chains having a molecular weight less than 8 kD.
The anticoagulant effect of UFH is mediated by interactions with anti-thrombin (AT), a serine protease inhibitor of thrombin. Binding of UFH to AT activates and accelerates inhibition by AT of the coagulation proteases thrombin and Factor Xa. It is estimated that about one third of hospitalized patients or 12 million patients per year receive UFH and LMWHs for a wide range of surgical and therapeutic procedures. However, about 1-5% of patients injected with heparin develop the disorder “heparin-induced thrombocytopenia” (HIT).
HIT is an adverse reaction to heparin, in which affected patients produce platelet-activating antibodies that bind complexes of platelet-factor 4 (PF4) and heparin (HIT antibodies), resulting in a prothrombotic and thrombocytopenic condition that in severe cases can be life-threatening. As many as 600,000 people per year develop HIT, which is double the number of breast cancer cases diagnosed annually in the United States (US Cancer Statistics Working Group), and nearly equal to the number of new cases of angina diagnosed each year (Go 2013). Failure to diagnose HIT can lead to catastrophic thrombosis if heparin therapy is continued. It is therefore important that a quick and accurate diagnosis of HIT is made when it occurs. An accurate diagnosis of HIT requires attention to both clinical findings and laboratory test results.
Conventional treatment for patients suspected of having HIT includes the immediate cessation of all heparin followed by prompt administration of a non-heparin parenteral anticoagulant (Cuker 2012). Thus, a rapid and reliable serologic test capable of detecting HIT antibodies and fully characterizing their potency and molecular properties can be extremely helpful to a clinician who is called upon to make these management decisions. One widely used immunologic assay is the PF4/heparin ELISA (PF4 ELISA), in which PF4 in a complex with UFH or another high molecular weight polyanion is bound to the wells of a plastic microtiter plate to serve as antigen. After incubation with highly diluted patient's serum, which potentially contains the antibodies of interest, and washing of the well, the presence of bound antibodies is detected with an enzyme-labeled anti-human immunoglobulin reagent.
The PF4 ELISA has a sensitivity for antibody detection approaching 100%, but a low positive predictive value for HIT diagnosis. Further, the ELISA is time consuming, costly and thus is normally not performed for single determinations but used to assay multiple samples, thus delaying the diagnosis process.
A recognized shortcoming of PF4 ELISA testing is that some patient serum samples that do not contain clinically significant HIT antibodies produce positive reactions in the PF4 ELISA (Whitlach 2010). To distinguish these false-positive reactions from true positives, it is a common practice to test patient samples in two wells, adding high dose heparin (typically 100 U/mL) to the second well. A true positive reaction will be inhibited by high dose heparin but a false positive will not, enabling the distinction between the two types of reactions to be made. The need for a high dose heparin step doubles the cost of PF4 ELISA testing.
An alternative method to confirm the presence of HIT antibodies is to use a functional assay such as the 14C-serotonin release assay (SRA), in which a positive test result correlates better with the clinical picture of HIT. The SRA involves incubation of patient's serum and various doses of UFH with washed platelets loaded with radioactive 14C-labeled serotonin. Sera from m patients with HIT having IgG antibodies form immune complexes with PF4/heparin, and the Fc portion of IgG incorporated into these complexes engages FcγRIIa (CD32) receptors on the labeled platelets, causing platelet activation and release of 14C-serotonin that can be measured in a beta counter instrument. The SRA is a technically demanding and expensive test performed routinely in only a few specialty laboratories. It is therefore impractical to perform the SRA on a timely basis when HIT is suspected and antibody detection is urgently needed to aid in patient management decisions. In addition, the high cost of the SRA, its limited availability, and the requirement for use of radioactivity make it less practical from a laboratory perspective than other approaches, particularly the invention described herein.
Most HIT antibodies are of the IgG isotype. However, IgA antibodies are common and IgM antibodies are not rare (Suh 1997, Greinacher 2007). It has been claimed by some that only IgG antibodies cause clinically significant HIT and that IgA and IgM antibodies need not be detected (Linhoff-Last 2001, Warkentin 2003). However, others have reported that HIT antibodies of the IgM and IgA isotypes can cause severe disease (Amiral 2006, Davoren 2006). Until this controversy is settled, it is important that test platforms be available that can distinguish between the three Ig isotypes. To achieve this using the standard PF4 ELISA format requires that three wells be utilized for each test, one each for secondary antibodies specific for IgG, IgA and IgM. If the high dose heparin inhibition step is added, a total of six wells would be needed for each test, greatly increasing the cost and turn-around time of each patient result. In part for this reason, testing for all three HIT antibody isotypes is not widely available currently.
Despite the high sensitivity of the PF4 ELISA and high specificity of the SRA, the shortcomings of these testing methods described above indicate a need for an assay capable of detecting HIT antibodies of the IgG, IgA and IgM isotypes, preferably rapidly and at low cost and preferably in a single reaction mixture.